EpiCompare Report
Sera Choi
EpiCompare compares different epigenetic datasets for quality control and benchmarking purposes. The report is divided into three sections:
- General Metrics: Metrics on fragments (duplication rate) and peaks (blacklisted peaks and peak widths) of input files
- Peak Overlap: Percentage and statistical significance of overlapping and unon-overlapping peaks.
- Functional Annotation: Functional annotation (ChromHMM, ChIPseeker and enrichment analysis) of peaks.
Input Datasets
- Reference peakfile: ENCODE_H3K27ac
- Total of 12 peak files:
## [1] "File1: H3K27ac_CnT_ActiveMotif_MACS2"
## [1] "File2: H3K27ac_CnT_ActiveMotif_SEACR"
## [1] "File3: H3K27ac_CnT_Abcam-ab4729_MACS2"
## [1] "File4: H3K27ac_CnT_Abcam-ab4729_SEACR"
## [1] "File5: H3K27ac_CnT_SRR8383507_MACS2"
## [1] "File6: H3K27ac_CnT_SRR8383507_SEACR"
## [1] "File7: H3K27me3_ENCODE_MACS2.bed"
## [1] "File8: H3K27me3_ENCODE_SEACR.bed"
## [1] "File9: H3K27ac_ENCODE"
## [1] "File10: H3K27ac_TIP_Abcam.phase_1_05_jan_2022.S_1_R1"
## [1] "File11: H3K27ac_TIP_Abcam.phase_1_05_jan_2022.S_1_R2"
## [1] "File12: H3K27ac_TIP_Abcam.phase_2_03_feb_2022.S_4_R1"Code
EpiCompare(peakfiles = list(H3K27ac_CnT_ActiveMotif_MACS2, H3K27ac_CnT_ActiveMotif_SEACR, H3K27ac_CnT_Abcam-ab4729_MACS2, H3K27ac_CnT_Abcam-ab4729_SEACR, H3K27ac_CnT_SRR8383507_MACS2, H3K27ac_CnT_SRR8383507_SEACR, H3K27me3_ENCODE_MACS2.bed, H3K27me3_ENCODE_SEACR.bed, H3K27ac_ENCODE, H3K27ac_TIP_Abcam.phase_1_05_jan_2022.S_1_R1, H3K27ac_TIP_Abcam.phase_1_05_jan_2022.S_1_R2, H3K27ac_TIP_Abcam.phase_2_03_feb_2022.S_4_R1),
blacklist = blacklist,
picard_files = list(),
reference = ENCODE_H3K27ac,
stat_plot = TRUE,
chrmHMM_plot = TRUE,
chrmHMM_annotation = "K562",
chipseeker_plot = TRUE,
enrichment_plot = TRUE,
interact = TRUE,
save_output = FALSE,
output_dir = "/Users/serachoi/Documents/EpiCompare")1. General Metrics
Peak Information
Column Description:
PeakN before tidy: Total number of peaks including those blacklisted and those in non-standard chromosomes.
Blacklisted peaks removed (%): Percentage of blacklisted peaks present in the sample. ENCODE blacklist includes regions in the hg19 genome that have anomalous and/or unstructured signals independent of the cell-line or experiment.
Non-standard peaks removed (%): Percentage of peaks identified in non-standard and/or mitochondrial chromosomes. Identified using
BRGenomics::tidyChromosomes().
PeakN after tidy: Total number of peaks after filtering blacklisted peaks and those in non-standard chromosomes.
NB: All analyses in EpiCompare are conducted on tidied datasets (i.e. blacklisted peaks and those in non-standard chromosomes removed)
| Sample | PeakN before tidy | Blacklisted peaks removed (%) | Non-standard peaks removed (%) | PeakN after tidy |
|---|---|---|---|---|
| H3K27ac_CnT_ActiveMotif_MACS2 | 2526 | 23.200 | 7.050 | 1762 |
| H3K27ac_CnT_ActiveMotif_SEACR | 3211 | 17.800 | 4.390 | 2497 |
| H3K27ac_CnT_Abcam-ab4729_MACS2 | 26077 | 6.800 | 0.874 | 24076 |
| H3K27ac_CnT_Abcam-ab4729_SEACR | 13530 | 5.690 | 0.761 | 12657 |
| H3K27ac_CnT_SRR8383507_MACS2 | 13456 | 3.470 | 0.498 | 12922 |
| H3K27ac_CnT_SRR8383507_SEACR | 6669 | 3.420 | 0.390 | 6415 |
| H3K27me3_ENCODE_MACS2.bed | 89900 | 2.850 | 0.414 | 86965 |
| H3K27me3_ENCODE_SEACR.bed | 107569 | 2.930 | 0.435 | 103953 |
| H3K27ac_ENCODE | 51176 | 0.952 | 0.000 | 50689 |
| H3K27ac_TIP_Abcam.phase_1_05_jan_2022.S_1_R1 | 60064 | 2.670 | 0.526 | 58143 |
| H3K27ac_TIP_Abcam.phase_1_05_jan_2022.S_1_R2 | 110709 | 2.530 | 0.447 | 107413 |
| H3K27ac_TIP_Abcam.phase_2_03_feb_2022.S_4_R1 | 73765 | 2.620 | 0.493 | 71472 |
Fragment Information
Metrics on fragments is shown only if Picard summary is provided. See manual for help.
Column Description:
- Mapped_Fragments: Number of mapped read pairs in the file.
- Duplication_Rate: Percentage of mapped sequence that is marked as duplicate.
- Unique_Fragments: Number of mapped sequence that is not marked as duplicate.
Peak widths
Distribution of peak widths in each sample.
2. Peak Overlap
Percentage Overlap
Heatmap of percentage of overlapping peaks between samples. Hover over the heatmap for percentage values.
N.B. How to interpret heatmap: [Samples in x-axis of heatmap] peaks in [Samples in y-axis of heatmap] peaks
Statistical Significance
The plot is shown only if a reference peak file is provided and
stat_plot = TRUE. Depending on the format of the reference
file, EpiCompare outputs different plots:
- Reference dataset has BED6+4 format (peakcalling performed with MACS2): EpiCompare generates paired boxplot per sample showing the distribution of -log10(q-value) of reference peaks that are overlapping and non-overlapping with the sample dataset.
- Reference dataset does not have BED6+4 format: EpiCompare generates a barplot of percentage of overlapping sample peaks with the reference, coloured by statistical significance (adjusted p-value) of the overlap.
Keys:
Reference peakfile: ENCODE_H3K27ac
- Overlap: Sample peaks in Reference peaks
- Unique: Sample peaks not in Reference peaks
3. Functional Annotation
3.1 ChromHMM
ChromHMM annotates and characterises peaks into different chromatin states. ChromHMM annotations used in EpiCompare were obtained from here.
- Cell-type annotation file used in this analysis: K562
All samples
ChromHMM annotation of individual samples.
Overlap: Sample peaks in Reference peaks
Percentage of Sample peaks found in Reference peaks (Reference peakfile: ENCODE_H3K27ac)
| Percentage | |
|---|---|
| H3K27ac_CnT_ActiveMotif_MACS2 | 63.200 |
| H3K27ac_CnT_ActiveMotif_SEACR | 77.200 |
| H3K27ac_CnT_Abcam-ab4729_MACS2 | 78.700 |
| H3K27ac_CnT_Abcam-ab4729_SEACR | 89.700 |
| H3K27ac_CnT_SRR8383507_MACS2 | 83.300 |
| H3K27ac_CnT_SRR8383507_SEACR | 92.500 |
| H3K27me3_ENCODE_MACS2.bed | 1.310 |
| H3K27me3_ENCODE_SEACR.bed | 0.731 |
| H3K27ac_ENCODE | 100.000 |
| H3K27ac_TIP_Abcam.phase_1_05_jan_2022.S_1_R1 | 8.250 |
| H3K27ac_TIP_Abcam.phase_1_05_jan_2022.S_1_R2 | 7.970 |
| H3K27ac_TIP_Abcam.phase_2_03_feb_2022.S_4_R1 | 9.730 |
ChromHMM annotation of sample peaks found in reference peaks.
Overlap: Reference peaks in Sample peaks
Percentage of Reference peaks found in Sample peaks (Reference peakfile: ENCODE_H3K27ac)
| Percentage | |
|---|---|
| H3K27ac_CnT_ActiveMotif_MACS2 | 2.24 |
| H3K27ac_CnT_ActiveMotif_SEACR | 4.65 |
| H3K27ac_CnT_Abcam-ab4729_MACS2 | 44.40 |
| H3K27ac_CnT_Abcam-ab4729_SEACR | 46.30 |
| H3K27ac_CnT_SRR8383507_MACS2 | 27.10 |
| H3K27ac_CnT_SRR8383507_SEACR | 25.30 |
| H3K27me3_ENCODE_MACS2.bed | 2.65 |
| H3K27me3_ENCODE_SEACR.bed | 1.83 |
| H3K27ac_ENCODE | 100.00 |
| H3K27ac_TIP_Abcam.phase_1_05_jan_2022.S_1_R1 | 12.70 |
| H3K27ac_TIP_Abcam.phase_1_05_jan_2022.S_1_R2 | 27.70 |
| H3K27ac_TIP_Abcam.phase_2_03_feb_2022.S_4_R1 | 23.00 |
ChromHMM annotation of reference peaks found in sample peaks.
Unique: Sample peaks not in Reference peaks
Percentage of sample peaks not found in reference peaks (Reference peakfile: ENCODE_H3K27ac)
| Percentage | |
|---|---|
| H3K27ac_CnT_ActiveMotif_MACS2 | 36.80 |
| H3K27ac_CnT_ActiveMotif_SEACR | 22.80 |
| H3K27ac_CnT_Abcam-ab4729_MACS2 | 21.30 |
| H3K27ac_CnT_Abcam-ab4729_SEACR | 10.30 |
| H3K27ac_CnT_SRR8383507_MACS2 | 16.70 |
| H3K27ac_CnT_SRR8383507_SEACR | 7.48 |
| H3K27me3_ENCODE_MACS2.bed | 98.70 |
| H3K27me3_ENCODE_SEACR.bed | 99.30 |
| H3K27ac_ENCODE | 0.00 |
| H3K27ac_TIP_Abcam.phase_1_05_jan_2022.S_1_R1 | 91.70 |
| H3K27ac_TIP_Abcam.phase_1_05_jan_2022.S_1_R2 | 92.00 |
| H3K27ac_TIP_Abcam.phase_2_03_feb_2022.S_4_R1 | 90.30 |
ChromHMM annotation of sample peaks not found in reference peaks.
Unique: Reference peaks not in Sample peaks
Percentage of reference peaks not found in sample peaks (Reference peakfile: ENCODE_H3K27ac)
| Percentage | |
|---|---|
| H3K27ac_CnT_ActiveMotif_MACS2 | 97.8 |
| H3K27ac_CnT_ActiveMotif_SEACR | 95.4 |
| H3K27ac_CnT_Abcam-ab4729_MACS2 | 55.6 |
| H3K27ac_CnT_Abcam-ab4729_SEACR | 53.7 |
| H3K27ac_CnT_SRR8383507_MACS2 | 72.9 |
| H3K27ac_CnT_SRR8383507_SEACR | 74.7 |
| H3K27me3_ENCODE_MACS2.bed | 97.4 |
| H3K27me3_ENCODE_SEACR.bed | 98.2 |
| H3K27ac_ENCODE | 0.0 |
| H3K27ac_TIP_Abcam.phase_1_05_jan_2022.S_1_R1 | 87.3 |
| H3K27ac_TIP_Abcam.phase_1_05_jan_2022.S_1_R2 | 72.3 |
| H3K27ac_TIP_Abcam.phase_2_03_feb_2022.S_4_R1 | 77.0 |
ChromHMM annotation of reference peaks not found in sample peaks.
3.2 ChIPseeker
EpiCompare uses annotatePeak function in
ChIPseeker package to annotate peaks with the nearest gene
and genomic region where the peak is located. The peaks are annotated
with genes
taken from the annotations of human genome hg19 provided by
Bioconductor.
3.3 Functional Enrichment Analysis
EpiCompare performs KEGG pathway and GO enrichment analysis using
clusterProfiler. annotatePeak function in
ChIPseeker package is first used to assign peaks to nearest
genes and biological themes amongst the genes are identified using
ontologies (KEGG and GO). The peaks are annotated with genes
taken from the annotations of human genome hg19 provided by
Bioconductor.
KEGG
GO
4. Session Info
## R version 4.1.2 (2021-11-01)
## Platform: x86_64-apple-darwin17.0 (64-bit)
## Running under: macOS Big Sur 11.2
##
## Matrix products: default
## LAPACK: /Library/Frameworks/R.framework/Versions/4.1/Resources/lib/libRlapack.dylib
##
## locale:
## [1] en_GB.UTF-8/en_GB.UTF-8/en_GB.UTF-8/C/en_GB.UTF-8/en_GB.UTF-8
##
## attached base packages:
## [1] stats4 stats graphics grDevices utils datasets methods base
##
## other attached packages:
## [1] EpiCompare_0.99.0 org.Hs.eg.db_3.14.0 AnnotationDbi_1.56.2 IRanges_2.28.0 S4Vectors_0.32.3 Biobase_2.54.0 BiocGenerics_0.40.0
## [8] testthat_3.1.2 devtools_2.4.3 usethis_2.1.5
##
## loaded via a namespace (and not attached):
## [1] utf8_1.2.2 tidyselect_1.1.2 heatmaply_1.3.0
## [4] RSQLite_2.2.10 htmlwidgets_1.5.4 TSP_1.2-0
## [7] grid_4.1.2 BiocParallel_1.28.3 scatterpie_0.1.7
## [10] munsell_0.5.0 codetools_0.2-18 withr_2.4.3
## [13] colorspace_2.0-3 GOSemSim_2.20.0 TxDb.Hsapiens.UCSC.hg38.knownGene_3.14.0
## [16] filelock_1.0.2 highr_0.9 knitr_1.37
## [19] rstudioapi_0.13 ggsignif_0.6.3 DOSE_3.20.1
## [22] MatrixGenerics_1.6.0 labeling_0.4.2 GenomeInfoDbData_1.2.7
## [25] polyclip_1.10-0 seqPattern_1.26.0 bit64_4.0.5
## [28] farver_2.1.0 downloader_0.4 rprojroot_2.0.2
## [31] vctrs_0.3.8 treeio_1.18.1 generics_0.1.2
## [34] xfun_0.29 BiocFileCache_2.2.1 R6_2.5.1
## [37] GenomeInfoDb_1.30.1 graphlayouts_0.8.0 seriation_1.3.2
## [40] locfit_1.5-9.4 bitops_1.0-7 BRGenomics_1.6.0
## [43] cachem_1.0.6 fgsea_1.20.0 gridGraphics_0.5-1
## [46] DelayedArray_0.20.0 assertthat_0.2.1 BiocIO_1.4.0
## [49] scales_1.1.1 vroom_1.5.7 ggraph_2.0.5
## [52] enrichplot_1.14.2 gtable_0.3.0 processx_3.5.2
## [55] tidygraph_1.2.0 rlang_1.0.1 genefilter_1.76.0
## [58] splines_4.1.2 rtracklayer_1.54.0 rstatix_0.7.0
## [61] lazyeval_0.2.2 impute_1.68.0 broom_0.7.12
## [64] abind_1.4-5 yaml_2.3.5 reshape2_1.4.4
## [67] backports_1.4.1 GenomicFeatures_1.46.5 crosstalk_1.2.0
## [70] qvalue_2.26.0 clusterProfiler_4.2.2 tools_4.1.2
## [73] ggplotify_0.1.0 gridBase_0.4-7 ggplot2_3.3.5
## [76] ellipsis_0.3.2 gplots_3.1.1 jquerylib_0.1.4
## [79] RColorBrewer_1.1-2 sessioninfo_1.2.2 Rcpp_1.0.8
## [82] plyr_1.8.6 progress_1.2.2 zlibbioc_1.40.0
## [85] purrr_0.3.4 RCurl_1.98-1.6 ps_1.6.0
## [88] prettyunits_1.1.1 ggpubr_0.4.0.999 viridis_0.6.2
## [91] SummarizedExperiment_1.24.0 ggrepel_0.9.1 fs_1.5.2
## [94] magrittr_2.0.2 data.table_1.14.2 DO.db_2.9
## [97] matrixStats_0.61.0 pkgload_1.2.4 hms_1.1.1
## [100] patchwork_1.1.1 evaluate_0.15 xtable_1.8-4
## [103] XML_3.99-0.9 gridExtra_2.3 compiler_4.1.2
## [106] biomaRt_2.50.3 tibble_3.1.6 KernSmooth_2.23-20
## [109] crayon_1.5.0 shadowtext_0.1.1 htmltools_0.5.2
## [112] ggfun_0.0.5 tzdb_0.2.0 tidyr_1.2.0
## [115] geneplotter_1.72.0 aplot_0.1.2 DBI_1.1.2
## [118] tweenr_1.0.2 genomation_1.26.0 ChIPseeker_1.30.3
## [121] dbplyr_2.1.1 MASS_7.3-55 rappdirs_0.3.3
## [124] boot_1.3-28 car_3.0-12 Matrix_1.4-0
## [127] readr_2.1.2 brio_1.1.3 cli_3.2.0
## [130] parallel_4.1.2 igraph_1.2.11 GenomicRanges_1.46.1
## [133] pkgconfig_2.0.3 TxDb.Hsapiens.UCSC.hg19.knownGene_3.2.2 registry_0.5-1
## [136] GenomicAlignments_1.30.0 plotly_4.10.0 foreach_1.5.2
## [139] xml2_1.3.3 ggtree_3.2.1 annotate_1.72.0
## [142] bslib_0.3.1 webshot_0.5.2 XVector_0.34.0
## [145] yulab.utils_0.0.4 callr_3.7.0 stringr_1.4.0
## [148] digest_0.6.29 Biostrings_2.62.0 rmarkdown_2.11
## [151] fastmatch_1.1-3 tidytree_0.3.8 dendextend_1.15.2
## [154] restfulr_0.0.13 curl_4.3.2 Rsamtools_2.10.0
## [157] gtools_3.9.2 rjson_0.2.21 lifecycle_1.0.1
## [160] nlme_3.1-155 jsonlite_1.8.0 carData_3.0-5
## [163] desc_1.4.0 viridisLite_0.4.0 BSgenome_1.62.0
## [166] fansi_1.0.2 pillar_1.7.0 lattice_0.20-45
## [169] pkgbuild_1.3.1 KEGGREST_1.34.0 fastmap_1.1.0
## [172] httr_1.4.2 plotrix_3.8-2 survival_3.2-13
## [175] GO.db_3.14.0 remotes_2.4.2 glue_1.6.2
## [178] iterators_1.0.14 png_0.1-7 bit_4.0.4
## [181] ggforce_0.3.3 stringi_1.7.6 sass_0.4.0
## [184] blob_1.2.2 DESeq2_1.34.0 caTools_1.18.2
## [187] memoise_2.0.1 dplyr_1.0.8 ape_5.6-1